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Biomedical subjects

J M Carter

Publications and source records attributed to J M Carter.

At least 19 recordsLinked to original sources
PubMed1997

Merozoite surface protein-1 epitopes recognized by antibodies that inhibit Plasmodium falciparum merozoite dispersal.

Serum antibodies from malaria immune donors can inhibit merozoite dispersal by forming immune complexes through surface-accessible regions of membrane associated antigens. Such merozoite forms are referred to as immune clusters of merozoites (ICM). Antibodies dissociated from ICM of Plasmodium falciparum identify a restricted subset of antigens, including merozoite surface protein-1 (MSP-1). We performed epitope mapping by comparing the reactivity of whole immune sera and ICM-derived antibodies in immunoblotting assays, using fourteen overlapping recombinant MSP-1 fragments, and by ELISA, using each of the 1720 octapeptides encoded within MSP-1. Antibodies in immune sera reacted with thirteen recombinant fragments and hundreds of octapeptides, but antibodies derived from ICM reacted with only six recombinant fragments and twenty octapeptides. Recombinant fragment recognition by ICM-derived antibodies was delimited to three regions 150-200 residues long, with seven of the octapeptide epitopes also mapping to these regions. The octapeptides recognized most strongly by antibodies in whole serum corresponded to the degenerate repeats near the N-terminus of MSP-1, however, neither recombinant fragments, nor octapeptides containing these degenerate repeats, were recognized by ICM-derived antibodies. Compared to reactions with recombinant fragments, the reactions observed with octapeptides were weak and may represent low-affinity mimetopes or cross-reactions. Alternatively, they may represent reactions with a portion of an epitope assembled from more than one non-contiguous peptide. These results suggest that ICM-derived antibodies can be used to map surface-accessible epitopes on MSP-1 and that the recombinant fragments with which they react are appropriate candidates for further evaluation as components of a malaria vaccine.

Animals↗
PubMed1997

Identification of novel human WW domain-containing proteins by cloning of ligand targets.

A recently described protein module consisting of 35-40 semiconserved residues, termed the WW domain, has been identified in a number of diverse proteins including dystrophin and Yes-associated protein (YAP). Two putative ligands of YAP, termed WBP-1 and WBP-2, have been found previously to contain several short peptide regions consisting of PPPPY residues (PY motif) that mediate binding to the WW domain of YAP. Although the function(s) of the WW domain remain to be elucidated, these observations strongly support a role for the WW domain in protein-protein interactions. Here we report the isolation of three novel human cDNAs encoding a total of nine WW domains, using a newly developed approach termed COLT (cloning of ligand targets), in which the rapid cloning of modular protein domains is accomplished by screening cDNA expression libraries with specific peptide ligands. Two of the new genes identified appear to be members of a family of proteins, including Rsp5 and Nedd-4, which have ubiquitin-protein ligase activity. In addition, we demonstrate that peptides corresponding to PY and PY-like motifs present in several known signaling or regulatory proteins, including RasGAP, AP-2, p53BP-2 (p53-binding protein-2), interleukin-6 receptor-alpha, chloride channel CLCN5, and epithelial sodium channel ENaC, can selectively bind to certain of these novel WW domains.

Amino Acid Sequence↗
PubMed1997

Effects of particle exposure and particle-elicited inflammatory cells on mutation in rat alveolar epithelial cells.

To investigate mechanisms underlying development of lung adenomas and carcinomas in rats exposed to poorly soluble particles the relationships between particle exposure, inflammation and mutagenesis in rat alveolar type II cells were characterized. Rats were exposed to saline or saline suspensions of 10 and 100 mg/kg of alpha-quartz, carbon black or titanium dioxide by intratracheal instillation. Fifteen months after exposure, bronchoalveolar lavage (BAL) cells were characterized as to number and type and lung histopathology performed. The alveolar type II cells were isolated and cultured in 6 thioguanine (6TG) containing media to select for mutation in the hprt gene. The potential contribution of lung inflammatory cells to in vivo mutagenic responses, were evaluated by co-culturing BAL cells with the rat alveolar epithelial cell line, RLE-6TN for 24 h and the RLE-6TN cells selected for 6TG resistance. Neutrophilic inflammation was detected in all rats exposed to 10 and 100 mg/kg of alpha-quartz and carbon black and 100 mg/kg titanium dioxide; epithelial hyperplasia was observed in rats exposed to 10 and 100 mg/kg of alpha-quartz and 100 mg/kg carbon black. Hprt mutation frequency was increased in alveolar type II cells from rats exposed to 10 and 100 mg/kg of alpha-quartz, 100 mg/kg carbon black and 100 mg/kg titanium dioxide. In vitro exposure of RLE-6TN cells to BAL cells from rats treated with 10 and 100 mg/kg of alpha-quartz or 100 mg/kg carbon black increased hprt mutant frequency. Both macrophage and neutrophil enriched BAL cell populations were mutagenic to RLE-6TN cells, however, the mutagenic activity appeared greatest for neutrophils. Addition of catalase to BAL cell:RLE-6TN co-cultures inhibited the increase in hprt mutation frequency. These studies demonstrate exposure of rats to doses of particles producing significant neutrophilic inflammation is associated with increased mutation in rat alveolar type II cells. The ability of particle-elicited macrophages and neutrophils to exert a mutagenic effect on epithelial cells in vitro supports a role for these inflammatory cells in the in vivo mutagenic effects of particle exposure. The inhibition of BAL cell-induced mutations by catalase implies a role for cell-derived oxidants in this response.

Animals↗
PubMed1997

A qualitative and quantitative analysis of the entorhinal cortex in schizophrenia.

The entorhinal cortex (ERC) has been implicated in schizophrenia by a number of studies. There is anatomical observation of neuronal heterotopias in the rostral ERC, which is consistent with a hypothesis of neurodevelopmental abnormalities in this disease. In view of the significant cytoarchitectonic variation of the ERC throughout its rostro-caudal extent, we performed a detailed subareal analysis of the rostral two-thirds of the entorhinal cortex (ERCr) in 14 postmortem schizophrenic brains and 14 matched controls (mean ages of 48 and 47 respectively). This systematic evaluation included both a qualitative microscopic analysis of morphogenetic anomalies that would be consistent with neurodevelopmental pathology and quantitative measurements of total neuronal number, average neuronal density, laminar volume and laminar depth from the cortical surface in cytoarchitectonically matched subareas of schizophrenic and control brains. Parcellation of the entire ERC on the basis of cytoarchitectonic criteria identified five distinct regions, similar to those described in the macaque, except that in the human brain three of the regions were further divisible into two or three subareas, yielding nine distinct cellular compartments. Five rostral areas, prorhinal (Pr), lateral (28L), intermediate rostral and caudal (281r and 281c), and sulcal (28S), comprise the ERCr. Gross and microscopic examination of these subdivisions throughout the ERCr failed to reveal laminar disorganization in any of the schizophrenic brains. The brains also did not differ significantly with respect to total neuronal number, total volume and neuronal density per laminar and subareal subdivision, or laminar thickness per entorhinal subarea. However, neuronal number and density were reduced by 12-18% in Pr and 28L, suggesting that mild quantitative abnormalities may exist in the ERCr and might possibly be revealed in a larger sample of schizophrenic brains. We have failed to confirm previous reports of laminar disorganization in the ERCr in brains of patients with schizophrenia; to the extent that this region is implicated in schizophrenia, the structural changes are likely to consist of more subtle cellular disturbances.

Adult↗
PubMed1997

Cytokines and particle-induced inflammatory cell recruitment.

The inflammatory response is a key component of host defense. However, excessive or persistent inflammation can contribute to the pathogenesis of disease. Inflammation is regulated, in part, by cytokines, which are small, typically glycosylated proteins that interact with membrane receptors to regulate cellular processes such as proliferation, differentiation, and secretion. During the past 10 years studies in humans and experimental animals have demonstrated that a cytokine called tumor necrosis factor alpha (TNF-alpha) plays a key role in the initiation of inflammatory responses in the lung and other tissues, including inflammation resulting from inhalation of noxious particles. There is now compelling evidence that one of the pathways by which inhaled particles stimulate the recruitment and subsequent activation of inflammatory cells is through the activation of lung macrophages to release TNF-alpha. TNF-alpha then acts via paracrine and autocrine pathways to stimulate cells to release other cytokines known as chemokines, which are directly chemotactic to leukocytes and other cells that participate in inflammatory and wound healing responses. In addition to a TNF-alpha-mediated pathway, there is growing evidence that some particles such as quartz and crocidolite can directly activate lung epithelial cells to release chemokines such as macrophage inflammatory protein-2, cytokine-induced neutrophil chemoattractant, and interleukin-8. A direct stimulatory effect of particles on lung epithelium may represent an additional or alternate pathway by which inhaled particles may elicit inflammation in the lung. Recent studies have suggested that oxidative stress may be a component of the mechanism by which particles activate cytokine production in cells such as macrophages and epithelial cells. The contribution of oxidative stress to particle-induced cytokine gene expression appears to be mediated, at least in part, through activation of the transcription factor nuclear factor kappa B.

Animals↗
PubMed1996

Multi-well ELISA based on independent peptide antigens for antibody capture. Application to Lyme disease serodiagnosis.

Novel procedures for the use of peptides as antibody-capture reagents in the ELISA format have been investigated. Epitope sequences from known immunodominant antigens of Borrelia burgdorferi were selected by screening peptide libraries with sera from patients with Lyme disease. Several epitope peptides were synthesized and immobilized, separately, on the ELISA plate as haptens on bovine serum albumin. Based on a comparative analysis of serum samples, it appears that peptide antigens can be used as effectively as a whole cell lysate to discriminate between Lyme disease and non-Lyme disease sera, thus avoiding dependence on bacterial sonicates which vary from passage to passage. Further improvements in epitope design for enhancement of accuracy in serodiagnosis are discussed.

Amino Acid Sequence↗
PubMed1996

Pulmonary inflammatory, chemokine, and mutagenic responses in rats after subchronic inhalation of carbon black.

Chronic inhalation of carbon black can produce carcinomas in rat lungs. At present the mechanisms underlying the rat lung tumor response to carbon black are unknown, although a significant role for inflammation and cell proliferation has been postulated. To investigate the processes which may contribute to development of rat lung tumors after carbon black exposure, we characterized the effects of subchronic inhalation of carbon black by rats on mutagenesis in alveolar epithelial cells, pulmonary inflammation, inflammatory cytokine/growth factor expression, and lung histopathology. Briefly, rats were exposed for 6 hr/day, 5 days/week for up to 13 weeks to 1.1, 7.1, and 52.8 mg/m3 carbon black and the effects on the lung were characterized after 6.5 and 13 weeks of exposure and 3 and 8 months of recovery. Endpoints characterized after carbon black exposure included mutation in the hprt gene of alveolar epithelial cells, changes in bronchoalveolar lavage fluid markers of lung injury and inflammation, expression of mRNA for the chemokines, MIP-2 and MCP-1, and lung histopathology. Lung burdens of carbon black were also determined. After 13 weeks of exposure to 1.1, 7.1, and 52.8 mg/m3 carbon black, lung burdens were 354, 1826, and 7861 micrograms carbon black, respectively. The lung clearance of carbon black appeared impaired after exposure to 7.1 and 52.8 mg/m3 carbon black, with the effects being more pronounced at the higher exposure level. Subchronic inhalation of 1.1 mg/m3 carbon black did not elicit any detectable adverse lung effects. A significant increase in hprt mutation frequency in alveolar epithelial cells was detected immediately after 13 weeks of exposure to 7.1 and 52.8 mg/m3 carbon black as well as after 3- and 8-month recovery periods for the group exposed to 52.8 mg/m3. No increase in hprt mutation frequency was observed for epithelial cells obtained from rats exposed to 1.1 mg/m3 carbon black. The observation that genotoxic effects (i.e., mutations) on alveolar epithelial cells occurred only after carbon black exposures which resulted in significant inflammation and epithelial hyperplasia supports the hypothesis that inflammatory cell-derives oxidants and increased cell proliferation play a role in the pathogenesis of rat lung tumors in response to carbon black.

Administration, Inhalation↗
PubMed1996

Binding properties of SH3 peptide ligands identified from phage-displayed random peptide libraries.

Combinatorial libraries have yielded high-affinity ligands for SH3 domains of a number of different proteins. We have shown that synthetic peptides containing these SH3 ligand sequences serve as specific probes of SH3 domains. Direct binding of the N-terminal biotinylated peptide ligands was conveniently detected in ELISA, filter-blotting, and dot-blotting experiments with the use of streptavidin-conjugated enzymes. In some cases, detection of peptide-SH3 interactions required that the biotinylated peptides first were preconjugated with streptavidin to form a multivalent complex. Interestingly, these nominally tetravalent SH3 peptide ligands cross-react to varying degrees with different SH3 domains. We have used such complexes to screen lambda cDNA expression libraries and have isolated clones that encode both known and novel SH3-domain-containing proteins. Based on the success of this methodology, we propose a general strategy by which ligands of a modular domain-containing protein can be isolated from random peptide libraries and used to screen cDNA expression libraries systematically for novel modular domain-containing proteins.

Amino Acid Sequence↗
PubMed1996

Transient effects of low-energy CO2 laser irradiation on dentinal impedance: implications for treatment of hypersensitive teeth.

This study evaluated the effect of CO2 laser irradiation on dentinal impedance by passing known cyclic potentials across dentinal wafers mounted as a window in an electrolytic cell and measuring the resulting electrical impedance. Wafers were equilibrated in 0.1 M of KCl. The wafer specimens were irradiated with a CO2 laser (12 W, 0.1 ms, energy density 1.25 J/cm2). The time for impedance equilibration after irradiation was compared with equilibration after mounting. Mounted samples required 48 h to approach equilibrium in the electrolyte. After laser irradiation, impedance of previously equilibrated samples also required 48 h to equilibrate. This, along with exponential curve fitting, confirmed that laser treatment reintroduced a transient alteration in impedance. Equilibration time after irradiation and the mounting were similar. Dentin desociation apparently caused this transient impedance. Energy dispersive X-ray analysis confirmed the disappearance of K+Cl- after irradiation. Therefore, laser irradiation may cause dentinal desociation, yielding temporary clinical relief of dentinal hypersensitivity until rehydration occurs.

Carbon Dioxide↗
PubMed1996

Enforcement of drunken driving laws in cases involving injured intoxicated drivers.

STUDY OBJECTIVE: To determine the frequency of driving while impaired (DWI) charges among alcohol-intoxicated drivers injured in motor vehicle crashes (MVCs) and any differences in the group of those charged compared with those not charged. METHODS: We performed a retrospective analysis of linked data from medical and judicial sources. Our setting was an urban emergency department of a trauma center serving a population of 1 million. We studied consecutive drivers injured in MVCs over a period of 15 months who had measured serum ethanol (BAC) levels of 100 mg/dL or higher. BAC, Trauma Score (TS), demographics, and crash data were linked to court records of charges, outcome, and prior convictions. The group of individuals who were charged with DWI were compared with those who were not charged. RESULTS: One hundred eighty-seven patients were studied; 53 (28%) were charged with DWI, and 32 (17% of total) were convicted. Two (7%) of 29 patients with severe injuries, 9 (28%) of 32 with moderate injuries, and 42 (33%) of 126 with nonsevere injuries were charged (P = .004). Eighteen (16%) of 112 patients with no prior convictions were charged; 20 (56%) of 36 patients with one, 11 (52%) of 21 with two, 3 (25%) of 12 with three, and 0 of 5 with four or more prior DWI convictions were charged (P < .001). There were no significant differences in BAC, demographics, or other measures between the two groups. CONCLUSION: Alcohol-impaired drivers who require ED treatment for injuries sustained in an MVC are infrequently charged with DWI. The likelihood of a DWI charge diminishes with increasing severity of injury. Repeat offenders are charged more often, but the frequency of charges does not increase with increasing number of prior DWI convictions.

Accidents, Traffic↗
PubMed1996

Apparent idiopathic hyponatremia in an ambulatory geriatric population.

OBJECTIVES: The purpose of this descriptive analysis is to demonstrate that among older patients with hyponatremia, there is a subset with apparent hyponatremia of the syndrome of inappropriate antidiuretic hormone secretion (SIADH) type, which appears associated with the aging process. A retrospective chart review was done to identify patients with hyponatremia and to classify them into non-SIADH, SIADH, and idiopathic categories. DESIGN: The participants were the ambulatory population of The Mount Sinai Hospital's outpatient geriatric clinic. Pertinent data generated during the antecedent 24 months were collected from outpatient charts and included sets of laboratory values (consisting of one sodium, glucose, BUN and creatinine level), age, sex, race, clinical history, present diagnosis, medications, and prescribed diets for each patient in the total population. RESULTS: Patients demonstrating hyponatremia with serum sodium levels of less than 135 mEq/L were identified from the total population and were further divided into SIADH and non-SIADH etiologies. The SIADH patients were then subdivided into known etiology SIADH and apparent idiopathic SIADH. In an ambulatory geriatric population of 405 subjects, 46 had hyponatremia, with SIADH the apparent cause in 27. Of the SIADH population, seven patients were considered to have idiopathic SIADH. An apparent predilection for development of this syndrome was observed among non-black patients and the old old. CONCLUSIONS: The results support the hypothesis that aging is a risk factor for the development of SIADH-like hyponatremia in a subset of older patients who do not have an apparent underlying etiology. Thus, aging may be an independent etiology for the development of hyponatremia.

Age Distribution↗
PubMed1996

The intermediate filament cytoskeleton of the lens: an ever changing network through development and differentiation. A minireview.

In the eye lens, intermediate filament proteins form two morphologically distinct polymers, 10-nm intermediate-sized filaments and beaded filaments. Coincidently, the beaded filament polymer and the proteins filensin and CP49 are specific to lens fibre cells and are therefore excellent markers for fibre cell differentiation. In the adult lens, filensin and CP49 are maintained throughout all stages of lens fibre cell differentiation whilst vimentin is apparently lost at a specific stage from the deeper cortical fibres. The expression of CP49 and filensin is coincident with the presence of beaded filaments suggesting these proteins are filament components. In association with alpha-crystallin, CP49 and filensin form beaded filaments in vitro. During fibre cell differentiation, filensin and CP49 are post-translationally modified. In the case of filensin, proteolysis results in two functionally distinct fragment sets, one derived from the alpha-helical rod domain and the other from the C-terminal tail domain of filensin. It is proposed that both filensin and CP49 are critically involved in organising the cytoplasmic and plasma membrane domains of the fibre cell and therefore essential to the optical properties of the lens.

Animals↗
PubMed1996

Alpha-quartz-induced chemokine expression by rat lung epithelial cells: effects of in vivo and in vitro particle exposure.

Chemokines are chemotactic cytokines that can play a key role in leukocyte recruitment to sites of tissue injury or infection. Previous studies have demonstrated that exposure to alpha-quartz as well as other noxious particles increases chemokine gene expression in rat lung, although the cells responsible for chemokine expression and the mechanisms underlying this response have remained unclear. The present studies demonstrate that exposure of rats to alpha-quartz induced expression of mRNA for the chemokine macrophage-inflammatory protein (MIP)-2 in epithelial cells lining the terminal bronchioles and alveolar ducts as well as macrophages and alveolar type II cells in the more distal lung. Treatment of rats with an anti-MIP-2 antiserum before alpha-quartz exposure markedly attenuated neutrophilic infiltration of the lungs demonstrating an important role for MIP-2 in alpha-quartz-induced pulmonary inflammation. In vitro exposure of primary cultures of rat alveolar type II cells or the rat alveolar type II cell line RLE-6TN to tumor necrosis factor-alpha, endotoxin, or alpha-quartz increased mRNA for MIP-2 as well as the structurally and functionally similar chemokine cytokine-induced neutrophil chemoattractant but not the chemokine MIP-1 alpha. The alpha-quartz-induced increase in epithelial MIP-2 mRNA resulted, at least in part, from increased gene transcription and was associated with the release of active MIP-2 protein. Induction of RLE-6TN MIP-2 and cytokine-induced neutrophil chemoattractant mRNA expression was not unique to alpha-quartz, being also increased by crocidolite asbestus fibers but not by titanium dioxide or MMVF-10 glass fibers. These findings indicate that epithelial cells contribute to chemokine expression in rat lung after exposure to alpha-quartz and potentially other noxious particles and suggest that alpha-quartz-activated MIP-2 expression in vivo results, at least in part, from a direct action of the particles on the lung epithelium.

Animals↗
PubMed1995

Characterization of phage that bind plastic from phage-displayed random peptide libraries.

During routine screenings of random peptide libraries displayed at the N terminus of the pIII coat protein of M13 bacteriophage, clones were isolated that bound directly to the polystyrene (PS) surface used to immobilize the target protein. The plastic-binding phage (P-b phi) bind to both unblocked plastic (PS and polyvinyl chloride, PVC) and plastic blocked with bovine serum albumin (BSA) but require non-ionic detergent to bind to plastic blocked with milk. Comparison of the P-b phi to antibody-binding phage (Ab-b phi) indicates that similar numbers of phage particles are bound, but fewer P-b phi the recovered by acid elution. Sequence determination of the displayed peptides reveals they lack amino-acid sequence similarity yet are highly enriched for the Tyr and Trp residues. However, because not all phage that display peptides rich in Tyr and Trp residues bind to plastic, and other methods of screening random peptide libraries have identified different classes of plastic-binding peptides, the relative abundance of Tyr and Trp residues should not be considered diagnostic of plastic-binding. In summary, these results help characterize one of the most common methods used to screen random peptide libraries and suggest strategies to avoid isolating P-b phi. Furthermore, while it is generally believed that proteins bind to plastic by non-specific interactions, these results show that a bias in aa composition can exist.

Amino Acid Sequence↗
PubMed1995

Cloning, expression, and functional characterization of rat MIP-2: a neutrophil chemoattractant and epithelial cell mitogen.

Macrophage inflammatory protein-2 (MIP-2) is a member of a family of cytokines that play roles in inflammatory, immune, and wound healing responses. To clone the cDNA for rat MIP-2, RNA was isolated from the lungs of Fischer 344 rats after instillation of lipopolysaccharide. Reverse transcription-polymerase chain reaction was performed by using synthetic oligonucleotide primers designed from the mouse MIP-2 cDNA sequence. A cDNA containing the coding region of rat MIP-2 was cloned and sequenced. Comparison to the mouse MIP-2 cDNA demonstrated 90.3% homology at the nucleotide level and 86% homology at the amino acid level. The rat MIP-2 cDNA was expressed in Escherichia coli and protein evaluated for bioactivity. The recombinant rat MIP-2 was chemotactic for rat neutrophils but did not stimulate migration of rat alveolar macrophages or human peripheral blood eosinophils or lymphocytes. In addition, the recombinant rat MIP-2 and the related rat chemokine, KC/CINC stimulated proliferation of rat alveolar epithelial cells but not fibroblasts in vitro.

Amino Acid Sequence↗
PubMed1995

TNF alpha and increased chemokine expression in rat lung after particle exposure.

Macrophage inflammatory protein 2 (MIP-2) and CINC (Cytokine-Induced-Neutrophil-Chemoattractant) are members of the chemokine family of inflammatory and immunoregulatory cytokines. MIP-2 and CINC exhibit potent neutrophil chemotactic activity and are thought to be key mediators of inflammatory cell recruitment in response to tissue injury and infection. In the present studies, we examined the potential involvement of MIP-2 and CINC in particle-elicited inflammation in the rat lung and the role of TNF alpha in particle-induced chemokine expression. Acute intratracheal instillation exposure of F344 rats to alpha quartz or titanium dioxide was shown to markedly increase steady-state levels of MIP-2 and CINC mRNA in lung tissue; a response which was associated with a significant increase in neutrophils in the bronchoalveolar lavage fluid. Additional studies demonstrated that acute inhalation of crocidolite fibers by rats also induced increased MIP-2 and CINC expression. Since previous studies had demonstrated that TNF alpha stimulates MIP-2 and CINC expression in vitro and that particle exposure induces TNF alpha production in rat lung we examined the role of TNF alpha in alpha quartz-induced MIP-2 gene expression. We demonstrated that passive immunization of mice against TNF alpha markedly attenuated the increased lung MIP-2 mRNA seen in response to alpha quartz inhalation. Collectively, these findings suggest that the chemokines MIP-2 and CINC play a role in neutrophil recruitment to the rat lung after particle exposure and indicate that particle-induced expression of these chemokines is mediated, at least in part, by production of TNF alpha.

Animals↗
PubMed1995

In vitro studies on the assembly properties of the lens proteins CP49, CP115: coassembly with alpha-crystallin but not with vimentin.

A rapid one-step purification procedure for CP49, an intermediate filament protein found in the lens, is described using reverse-phase HPLC. This protein is one of the major intermediate filament proteins of the lens fibre cells and is found in both the water insoluble fraction (WIF) and the water soluble fraction (WSF) of the lens. In order to better understand the physiological role of CP49 in lens transparency we have purified CP49 from both compartments and compared the in vitro assembly characteristics of both by electron microscopy and sedimentation assays. Our studies showed that CP49, when mixed with another lens intermediate filament protein, CP115, forms 10 nm intermediate filaments. Vimentin, another intermediate filament protein found in the lens, was unable to coassemble with CP115, thus demonstrating the specificity of the interaction of CP49 with CP115. CP49 isolated from either the WIF or the WSF formed 10-nm filaments with CP115 and indicated that CP49 from both these lens cell compartments had similar in vitro assembly characteristics. This also suggested that the post-translational modifications observed for CP49 from the different compartments was of little apparent consequence to filament formation. The inability to reconstitute beaded filaments from CP49 and CP115 suggested that other lens proteins may be needed in the reconstitution assay before these lens specific cytoskeletal elements could be repolymerised from their purified protein components. CP49 and CP115 were therefore assembled in the presence of alpha-crystallins and a beaded filament structure was observed as has been seen with type III intermediate filament proteins assembled with alpha-crystallins.

Animals↗